Anatomy and Neuroscience - Research Publications
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ItemRho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid(ELSEVIER, 2013-05)We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA's effects on the expansion of neural stem/progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury.
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ItemNo Preview AvailableDirecting Human Induced Pluripotent Stem Cells into a Neurosensory Lineage for Auditory Neuron Replacement(MARY ANN LIEBERT, INC, 2014-08)Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.
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ItemHypothermia protects human neurons(SAGE PUBLICATIONS LTD, 2014-07)BACKGROUND AND AIMS: Hypothermia provides neuroprotection after cardiac arrest, hypoxic-ischemic encephalopathy, and in animal models of ischemic stroke. However, as drug development for stroke has been beset by translational failure, we sought additional evidence that hypothermia protects human neurons against ischemic injury. METHODS: Human embryonic stem cells were cultured and differentiated to provide a source of neurons expressing β III tubulin, microtubule-associated protein 2, and the Neuronal Nuclei antigen. Oxygen deprivation, oxygen-glucose deprivation, and H2 O2 -induced oxidative stress were used to induce relevant injury. RESULTS: Hypothermia to 33°C protected these human neurons against H2 O2 -induced oxidative stress reducing lactate dehydrogenase release and Terminal deoxynucleotidyl transferase dUTP nick end labeling-staining by 53% (P ≤ 0·0001; 95% confidence interval 34·8-71·04) and 42% (P ≤ 0·0001; 95% confidence interval 27·5-56·6), respectively, after 24 h in culture. Hypothermia provided similar protection against oxygen-glucose deprivation (42%, P ≤ 0·001, 95% confidence interval 18·3-71·3 and 26%, P ≤ 0·001; 95% confidence interval 12·4-52·2, respectively) but provided no protection against oxygen deprivation alone. Protection (21%) persisted against H2 O2 -induced oxidative stress even when hypothermia was initiated six-hours after onset of injury (P ≤ 0·05; 95% confidence interval 0·57-43·1). CONCLUSION: We conclude that hypothermia protects stem cell-derived human neurons against insults relevant to stroke over a clinically relevant time frame. Protection against H2 O2 -induced injury and combined oxygen and glucose deprivation but not against oxygen deprivation alone suggests an interaction in which protection benefits from reduction in available glucose under some but not all circumstances.
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ItemCharacterization of forebrain neurons derived from late-onset Huntington's disease human embryonic stem cell lines(FRONTIERS MEDIA SA, 2013-04-05)Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG37 and CAG51 typical late-onset repeat expansions in comparison to wildtype control lines during undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate-evoked responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.
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ItemStem Cells as In Vitro Models of Disease(HINDAWI LTD, 2012)
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ItemStimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells(BIOMED CENTRAL LTD, 2011-04-04)INTRODUCTION: Unrestricted somatic stem cells (USSC) derived from umbilical cord blood are an attractive alternative to human embryonic stem cells (hESC) for cellular therapy. USSC are capable of forming cells representative of all three germ line layers. The aim of this study was to determine the potential of USSC to form definitive endoderm following induction with Activin A, a protein known to specify definitive endoderm formation of hESC. METHODS: USSC were cultured for (1) three days with or without 100 ng/ml Activin A in either serum-free, low-serum or serum-containing media, (2) three days with or without 100 ng/ml Activin A in combination with 10 ng/ml FGF4 in pre-induction medium, or (3) four days with or without small molecules Induce Definitive Endoderm (IDE1, 100 nM; IDE2, 200 nM) in serum-free media. Formation of definitive endoderm was assessed using RT-PCR for gene markers of endoderm (Sox17, FOXA2 and TTF1) and lung epithelium (surfactant protein C; SPC) and cystic fibrosis transmembrane conductance regulator; CFTR). The differentiation capacity of Activin A treated USSC was also assessed. RESULTS: Activin A or IDE1/2 induced formation of Sox17+ definitive endoderm from hESC but not from USSC. Activin A treated USSC retained their capacity to form cells of the ectoderm (nerve), mesoderm (bone) and endoderm (lung). Activin A in combination with FGF4 did not induce formation of Sox17+ definitive endoderm from USSC. USSC express both Activin A receptor subunits at the mRNA and protein level, indicating that these cells are capable of binding Activin A. CONCLUSIONS: Stimulation of the Nodal signaling pathway with Activin A or IDE1/2 is insufficient to induce definitive endoderm formation from USSC, indicating that USSC differ in their stem cell potential from hESC.
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ItemGli1 Is an Inducing Factor in Generating Floor Plate Progenitor Cells from Human Embryonic Stem Cells(WILEY-BLACKWELL, 2010-10)Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer.
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ItemFunctional Characterization of Friedreich Ataxia iPS-Derived Neuronal Progenitors and Their Integration in the Adult Brain(PUBLIC LIBRARY SCIENCE, 2014-07-07)Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function. Furthermore, FA iPS-derived neural progenitors are able to differentiate into functional neurons and integrate in the nervous system when transplanted into the cerebellar regions of host adult rodent brain. These are the first studies to describe both in vitro and in vivo characterization of FA iPS-derived neurons and demonstrate their capacity to survive long term. These findings are highly significant for developing FRDA therapies using patient-derived stem cells.
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ItemA reduction in Npas4 expression results in delayed neural differentiation of mouse embryonic stem cells(BMC, 2014-05-08)INTRODUCTION: Npas4 is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. It is known that in the adult brain Npas4 plays an important role in several key aspects of neurobiology including inhibitory synapse formation, neuroprotection and memory, yet very little is known about the role of Npas4 during neurodevelopment. The aim of this study was to examine the expression and function of Npas4 during nervous system development by using a combination of in vivo experiments in the developing mouse embryo and neural differentiation of embryonic stem cells (ESCs) as an in vitro model of the early stages of embryogenesis. METHODS: Two different neural differentiation paradigms were used to investigate Npas4 expression during neurodevelopment in vitro; adherent monolayer differentiation of mouse ESCs in N2B27 medium and Noggin-induced differentiation of human ESCs. This work was complemented by direct analysis of Npas4 expression in the mouse embryo. The function of Npas4 in the context of neurodevelopment was investigated using loss-of-function experiments in vitro. We created several mouse ESC lines in which Npas4 expression was reduced during neural differentiation through RNA interference and we then analyzed the ability of these Npas4 knockdown mouse ESCs lines to undergo neural differentiation. RESULTS: We found that while Npas4 is not expressed in undifferentiated ESCs, it becomes transiently up-regulated during neural differentiation of both mouse and human ESCs at a stage of differentiation that is characterized by proliferation of neural progenitor cells. This was corroborated by analysis of Npas4 expression in the mouse embryo where the Npas4 transcript was detected specifically in the developing forebrain beginning at embryonic day 9.5. Finally, knockdown of Npas4 expression in mouse ESCs undergoing neural differentiation affected their ability to differentiate appropriately, resulting in delayed neural differentiation. CONCLUSIONS: Here we provide the first evidence that Npas4 is expressed during embryonic development and that it may have a developmental role that is unrelated to its function in the adult brain.
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ItemMultipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System(WILEY, 2015-06)The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.